The G Protein-coupled receptors (GPCRs) are a large superfamily of seven-helix transmembrane receptors that mediate a wide range of cellular functions, and are implicated in numerous human diseases including cardiovascular disease, chronic pain, cancer, metabolic disorders, central nervous system disorders, asthma, hypertension, and schizophrenia. GPCRs transmit information from the outside of a cell to the inside, signaling the cell to respond to changes in its environment. They represent the largest and richest class of drug targets: over half of all drugs marketed target a GPCR, including familiar drug classes such as antihypertensives, antihistamines, antacids, and anxiety control medications. Because of their importance, most pharmaceutical companies maintain large programs aimed at these receptors, and resources are increasingly being devoted to GPCRs, particularly since thousands have been identified from genomic sequencing. However, little is known about the structural and functional features of GPCRs. Moreover, while the principles governing stability and folding of soluble proteins are fairly well understood, little is known about how those principles map onto integral membrane proteins, which represent 30% of the human genome. The Robinson group is currently investigating conserved and variable features of structure, function, folding, and assembly of GPCRs, which are the largest family of integral membrane proteins. Our focus is on obtaining novel structural and conformational information, and using protein engineering approaches to produce major advances in the study of GPCR structural biology, biochemistry, and biophysics. We have developed new insights into determinants of structure, stability, and ligand recognition of this vitally important class of proteins. In a model GPCR, the beta2 adrenergic receptor (beta2AR), we have identified a folding intermediate that is a core functional domain, and an alternate folding pathway that leads to an inactive state. The goal of this project is to understand the assembly pathways for beta2AR, and identify amino acid determinants of stability for the native, intermediate, and inactive states. Our unique interdisciplinary approach combines the use of site-directed mutagenesis with detailed biophysical studies of the equilibrium and kinetic properties of these receptors. We will also synthesize and characterize peptide fragments of the receptor to elucidate the hierarchy of recognition events that drive the receptor assembly process. In future studies, we will use experimental and bioinformatics tools to extend these principles to other receptors, including closely related members of the same subfamily, other subfamilies, and the entire superfamily. We know of no analogous studies of this important receptor superfamily.